Patchwork peptide sequencing: extraction of sequence information from accurate mass data of peptide tandem mass spectra recorded at high resolution

The accurate mass values of all immonium, y(1), y(2), a(2), and b(2) ions of tryptic peptides composed of the 20 standard amino acids were calculated. The differences between adjacent masses in this data set are greater than 10 mDa for more than 80% of the values. Using this mass list, the majority of low mass ions in quadrupole-time of flight tandem mass spectra of peptides from tryptic digests and from an elastase digest could be assigned. Besides the a(2)/b(2) ions, which carry residues 1-2 from the N-terminus, a variety of internal dipeptide b ions were regularly observed. In case internal proline was present, corresponding dipeptide b ions carrying proline at the N-terminal position occurred. By assigning the dipeptide b ions on the basis of their accurate mass, bidirectional or unidirectional sequence information was obtained, which is localized to the peptide N-terminus (a(2)/b(2) ions) or not localized (internal b ions). Identification of the y(1) and y(2) ions by their accurate mass provides unidirectional sequence information localized to the peptide C-terminus. It is shown that this patchwork-type sequence information extractable from accurate mass data of low-mass ions is highly efficient for protein identification.     

The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.     

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.     

Elevation of Taurine in Hippocampal Extracellular Fluid and Cerebrospinal Fluid of Acutely Hypoosmotic Rats: Contribution by Influx from Blood?

Previous work has demonstrated that there is a selective increase in extracellular taurine in the brain during acute water intoxication. One aim of the present study was to investigate whether plasma taurine contributes to this increase. To this end, the concentrations of taurine, other amino acids, and ethanolamine (EA) were measured in plasma and CSF of urethane-anesthetized rats injected with 150 ml/kg body weight of distilled water. Blood pressure, blood gases, and pH, as well as plasma and CSF osmolality, were also measured. The CSF level of albumin was quantitated to study the function of the blood-CSF barrier. In separate experiments, hippocampal microdialysis was performed to determine the effects of acute plasma hypoosmolality on extracellular amino acids. Finally, the effect of water injection on hippocampal specific gravity and tissue amino acids was assessed. Blood gases and pH were essentially unchanged after water administration. Mean arterial blood pressure increased to peak levels approximately 50 mm Hg above control. Plasma osmolality decreased rapidly, whereas the depression of CSF osmolality was slower and less pronounced. The average volume of the hippocampus increased by 8%. Water injection was accompanied by a 25-fold elevation of taurine in plasma, whereas phosphoethanolamine (PEA) and EA increased moderately. A small fraction of the increase in plasma taurine might derive from blood cells because dilution of blood in vitro led to doubled plasma levels of the amino acid. Taurine, PEA, and EA increased consistently in CSF and hippocampal microdialysates. Plasma hypoosmolality transiently opened the blood-CSF barrier is reflected by augmented CSF concentrations of albumin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of seasonal variation in longitudinal growth of height in a sample of boys from Stuttgart Carlsschule, 1771-1793, using combined principal component analysis and maximum likelihood principle

Recent progress in modelling individual growth has been achieved by combining the principal component analysis and the maximum likelihood principle. This combination models growth even in incomplete sets of data and in data obtained at irregular intervals. We re-analysed late 18th century longitudinal growth of German boys from the boarding school Carlsschule in Stuttgart. The boys aged 6-23 years, were measured at irregular 3-12 monthly intervals during the period 1771-1793. At the age of 18 years, mean height was 1652 mm, but height variation was large. The shortest boy reached 1474 mm, the tallest 1826 mm. Measured height closely paralleled modelled height, with mean difference of 4 mm, SD 7 mm. Seasonal height variation was found. Low growth rates occurred in spring and high growth rates in summer and autumn. The present study demonstrates that combining the principal component analysis and the maximum likelihood principle enables growth modelling in historic height data also.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.     

Fast corroding,thin magnesium coating displays antibacterial effects and low cytotoxicity

Bacterial colonisation and biofilm formation are characteristics of implant-associated infections. In search of candidates for improved prosthetic materials, fast corroding Mg-based coatings on titanium surfaces were examined for their cytotoxic and antimicrobial properties. Human osteoblasts and Staphylococcus epidermidis were each cultured on cylindrical Ti samples coated with a thin layer of Mg/Mg₄₅Zn₅Ca, applied via magnetron sputtering. Uncoated titanium samples served as controls. S. epidermidis was quantified by counting colony forming units. The biofilm-bound fraction was isolated via ultrasonic treatment, and the planktonic fraction via centrifugation. Biofilm-bound S. epidermidis was significantly decreased by approximately four to five orders of magnitude in both Mg- and Mg₄₅Zn₅Ca-coated samples after seven days compared to the control. The osteoblast viability was within the tolerance threshold of 70% stated in DIN EN ISO 10993-5:2009-10 for Mg (~80%) but not for Mg₄₅Zn₅Ca (~25%). Accordingly, Mg-coated titanium was identified as a promising candidate for an implant material with antibacterial properties and low cytotoxicity levels. The approach of exploiting fast corrosion contrasts with existing methods, which have generally focused on reducing corrosion.     

Root Morphology and Anchorage of Six Native Tree Species from a Tropical Montane Forest and an Elfin Forest in Ecuador

Root architecture of tree species was investigated at two different altitudes in tropical forests in Ecuador. Increasing altitude was accompanied by higher wind speeds and more shallow soils, while slope angles of both sites were comparable (20–50°). Three tree species typical for the montane forest at 1900 m (Graffenrieda emarginata (Ruiz & Pav.) Triana (Melastomataceae), Clethra revoluta (Ruiz & Pav.) Spreng. (Clethraceae), Vismia tomentosa Ruiz & Pav. (Clusiaceae)) and for the elfin forest at 3000 m (Weinmannia loxensis Harling (Cunoniaceae), Clusia spec. (Clusiacaea) Styrax foveolaria Perkins (Styraceae)) were examined. At 1900 m, 92% of the trees grew upright, in comparison to 52% at 3000 m. At 3000 m, 48% of the trees were inclined, lying or even partly uprooted. At this altitude, all trees with tap roots or with shoots connected by coarse rhizomes, 83% of the trees with stilt roots, and 50% of the trees in which stems or roots were supported by other trees grew upright, suggesting that these characteristics were relevant for tree stability. Root system morphology differed markedly between altitudes. In contrast to 1900 m, where 20% of structural roots originated in the deeper mineral soil, root origin at 3000 m was restricted to the forest floor. The mean ratio of root cross sectional area to tree height decreased significantly from 6.1 × 10⁻³ m² m⁻¹ at 1900 m to 3.2 × 10⁻³ m² m⁻¹ at 3000 m. The extent of root asymmetry increased significantly from 0.29 at 1900 m to 0.62 at 3000 m. This was accompanied by a significantly lower number of dominant roots at 3000 m (2.3 compared to 3.8 at 1900 m). In conclusion, native tree species growing in tropical montane and elfin forests show a variety of root traits that improve tree stability. Root system asymmetry is less important for tree stability where anchorage is provided by a deep and solid root–soil plate. When deep rooting is impeded, root traits improving the horizontal extension of the root–soil plate are more pronounced or occur more frequently. Furthermore, mutual mechanical support of roots and stems of neighboring trees seems to be an appropriate mechanism to provide anchorage in soils with low bulk density and in environments with high wind speeds.